Derivatives of 2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]pyrido[3,4b]-indole-1,4-dione

ABSTRACT

Compounds of the general structural formula (I) and use of the compounds and salts and solvates thereof, as therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATION

This is the U.S. national phase application of International ApplicationNo. PCT/US01/15936, filed May 15, 2001, which claims the benefit ofprovisional patent application Ser. No. 60/210,137, filed Jun. 7, 2000.

FIELD AND BACKGROUND OF THE INVENTION

This invention relates to a series of compounds, to methods of preparingthe compounds, to pharmaceutical compositions containing the compounds,and to their use as therapeutic agents. In particular, the PRESENTinvention relates to compounds that are potent and selective inhibitorsof cyclic guanosine 3′,5′-monophosphate specific phosphodiesterase(cGMP-specific PDE), in particular PDE5, and have utility in a varietyof therapeutic areas wherein such inhibition is considered beneficial,including the treatment of cardiovascular disorders and erectiledysfunction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compounds having the generalstructural formula (I):

wherein R¹ is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynyl, haloC₁₋₆alkyl, C₃₋₈cycloalkyl,heteroC₃₋₈cycloalkyl, C₃₋₈cycloalkylC₁₋₃alkyl, arylC₁₋₃-alkyl, andheteroarylC₁₋₃alkyl;

-   -   R² is selected from the group consisting of an optionally        substituted monocyclic aromatic ring selected from the group        consisting of benzene, thiophene, furan, and pyridine, and an        optionally substituted bicyclic ring        wherein the fused ring A is a 5- or 6-membered ring, saturated        or partially or fully unsaturated, and comprises carbon atoms        and optionally one or two heteroatoms selected from oxygen,        sulfur, and nitrogen;

R³ is selected from the group consisting of hydrogen and C₁₋₆alkyl,

or R¹ and R³ together represent a 3- or 4-membered alkyl or alkenylchain component of a 5- or 6-membered ring;

R⁴, independently, is selected from the group consisting of

aryl,

Het,

C₃₋₈cycloalkyl,

YC₃₋₈cycloalkyl (wherein Y is oxygen, sulfur, or NR^(a)),

C(═O)R^(a),

OC(═O)R^(a),

C(═O)OR^(a),

C₁₋₄alkyleneNR^(a)R^(b),

C₁₋₄alkyleneHet,

C₁₋₄alkyleneC(═O)OR^(a),

C(═O)NR^(a)SO₂R^(c),

C(═O)C₁₋₄alkyleneHet,

C(═O)NR^(a)R^(b),

C(═O)NR^(a)R^(c),

C(═O)NR^(a)C₁₋₄alkyleneOR^(b),

C(═O)NR^(a)C₁₋₄alkyleneHet,

OR^(a),

OC₁₋₄alkyleneC(═O)OR^(a),

OC₂₋₄alkyleneNR^(a)R^(b),

OC₁₋₄alkyleneHet,

OC₂₋₄alkyleneOR^(a),

OC₁₋₄alkyleneNR^(a)C(═O)OR^(b),

NR^(a)R^(b),

NR^(a)C₁₋₄alkyleneNR^(a)R^(b),

NR^(a)C(═O)R^(b),

NR^(a)C(═O)NR^(a)R^(b),

N(SO₂C₁₋₄alkyl)₂,

NR^(a)(SO₂C₁₋₄alkyl),

nitro (NO₂),

trifluoromethyl,

trifluoromethoxy,

cyano (CN),

SO₂NR^(a)R^(b),

SO₂R^(a),

SOR^(a),

SR^(a),

and OSO₂CF₃;

R⁵ is selected from the group consisting of hydrogen, halogen, andC₁₋₆alkyl;

or R⁴ and R⁵ are taken together with the carbon atoms to which they areattached to form a 5-, 6-, or 7-membered ring, saturated or partially orfully unsaturated, optionally substituted and optionally containing oneor two heteroatoms selected from the group consisting of oxygen, sulfur,and nitrogen;

R^(a) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₃₋₈cycloalkyl, aryl, arylC₁₋₃alkyl, C₁₋₃alkylenearyl, and Het;

R^(b) is selected from the group consisting of hydrogen, C₁₋₆alkyl,aryl, arylC₁₋₃alkyl, C₁₋₃alkylenearyl, and Het;

R^(c) is phenyl or C₄₋₆cycloalkyl, either optionally substituted withone or more substituents selected from the group consisting of halo,C(═O)OR^(a), and OR^(a);

Het is a 5- or 6-membered heterocyclic group, saturated or partially orfully unsaturated, containing at least one heteroatom selected from thegroup consisting of oxygen, nitrogen, and sulfur, and optionallysubstituted with C₁₋₄alkyl or C(═O)OR^(b);

q is 1, 2, or 3; and

pharmaceutically acceptable salts and hydrates thereof.

As used herein, the term “alkyl” includes straight chained and branchedhydrocarbon groups containing the indicated number of carbon atoms,typically methyl, ethyl, and straight chain and branched propyl andbutyl groups. The hydrocarbon group can contain up to 16 carbon atoms.The term “alkyl” includes “bridged alkyl,” i.e., a C₆-C₁₆ bicyclic orpolycyclic hydrocarbon group, for example, norbornyl, adamantyl,bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, ordeca-hydronaphthyl. The term “cycloalkyl” is defined as a cyclic C₃-C₈hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, andcyclopentyl.

“Heterocycloalkyl” is similarly defined as a ring containing three toeight atoms, with one to three atoms selected from the group consistingof oxygen, nitrogen, and sulfur, with the remaining atoms being carbon.

The terms “alkenyl” and “alkynyl” are defined identically as “alkyl,”except for containing a carbon-carbon double bond or carbon-carbontriple bond, respectively.

The term “alkylene” refers to an alkyl group having a substituent. Forexample, the term “C₁₋₃alkylenearyl” refers to an alkyl group containingone to three carbon atoms, and substituted with an aryl group. The term“alkenylene” as used herein is similarly defined, and contains theindicated number of carbon atoms and a carbon-carbon double bond, andincludes straight chained and branched alkenylene groups, likeethyenylene.

The term “halo” or “halogen” is defined herein to include fluorine,bromine, chlorine, and iodine.

The term “haloalkyl” is defined herein as an alkyl group substitutedwith one or more halo substituents, either fluoro, chloro, bromo, iodo,or combinations thereof. Similarly, “halocycloalkyl” is defined as acycloalkyl group having one or more halo substituents.

The term “aryl,” alone or in combination, is defined herein as amonocyclic or polycyclic aromatic group, preferably a monocyclic orbicyclic aromatic group, e.g., phenyl or naphthyl, that can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, halo, alkyl, hydroxy, hydroxyalkyl, alkoxy,alkoxyalkyl, haloalkyl, nitro, amino, alkylamino, acylamino, alkylthio,alkylsulfinyl, and alkylsulfonyl. Exemplary aryl groups include phenyl,naphthyl, tetra-hydronaphthyl, 2-chlorophenyl, 3-chlorophenyl,4-chlorophenyl, 2-methylphenyl, 4-methoxyphenyl,3-trifluoromethylphenyl, 4-nitrophenyl, and the like.

The term “heteroaryl” is defined herein as a monocyclic or bicyclic ringsystem containing one or two aromatic rings and containing at least onenitrogen, oxygen, or sulfur atom in an aromatic ring, and which can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, substituents, like halo, alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino,acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Examples ofheteroaryl groups include thienyl, furyl, pyridyl, oxazolyl, quinolyl,isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidizolyl,benzothiazolyl, pyrazinyl, pyrinidinyl, thiazolyl, and thiadiazolyl.

The term “alkoxyalkyl” is defined as an alkyl group wherein a hydrogenhas been replaced by an alkoxy group. The term “(alkylthio)alkyl” isdefined similarly as alkoxyalkyl, except a sulfur atom, rather than anoxygen atom, is present.

The term “hydroxy” is defined as —OH.

The term “alkoxy” is defined as —OR, wherein R is alkyl.

The term “hydroxyalkyl” is defined as a hydroxy group appended to analkyl group.

The term “amino” is defined as —NH₂, and the term “alkylamino” isdefined as —NR₂, wherein at least one R is alkyl and the second R isalkyl or hydrogen.

The term “acylamino” is defined as RC(═O)N, wherein R is alkyl or aryl.

The term “alkylthio” is defined as —SR, wherein R is alkyl.

The term “alkylsulfinyl” is defined as R—SO₂, wherein R is alkyl.

The term “alkylsulfonyl” is defined as R—SO₃, wherein R is alkyl.

The term “nitro” is defined as —NO₂.

The term “trifluoromethyl” is defined as —CF₃.

The term “trifluoromethoxy” is defined as —OCF₃.

The term “cyano” is defined as —CN.

In a preferred embodiment, R¹ is selected from the group consisting ofhydrogen, C₁₋₆alkyl, haloC₁₋₆alkyl, C₃₋₈cycloalkyl,C₃₋₈cycloalkylC₁₋₃alkyl, arylC₂₋₃alkyl, and heteroarylC₂₋₃alkyl.

In a preferred embodiment, R² is the optionally substituted bicyclicring system

wherein the bicyclic ring can represent, for example, naphthalene orindene, or a heterocycle, such as benzoxazole, benzothiazole,benzisoxazole, benzimidazole, quinoline, indole, benzothiophene, orbenzofuran, or

wherein n is an integer 1 or 2, and X, independently, are C(R^(a))₂, O,S, or NR^(a). The bicyclic ring comprising the R² substituent typicallyis attached to the rest of the molecule by a phenyl ring carbon atom.

In a preferred group of compounds of formula (I), R² is represented byan optionally substituted bicyclic ring

wherein n is 1 or 2, and X, independently, are CH₂ or O. Especiallypreferred R² substituents include

Within this particular group of compounds, nonlimiting examples ofsubstituents for the bicyclic ring include halogen (e.g., chlorine),C₁₋₃alkyl (e.g., methyl, ethyl, or i-propyl), OR^(a) (e.g., methoxy,ethoxy, or hydroxy), CO₂R^(a), halomethyl or halomethoxy (e.g.,trifluoromethyl or trifluoromethoxy), cyano, nitro, and NR^(a)R^(b).

In another preferred embodiment, R⁴ is selected from the groupconsisting of aryl, Het, OR^(a), C(═O)OR^(a), C₁₋₄alkyleneNR^(a)R^(b),OC(═O)R^(a), C(═O)R^(a), NR^(a)R^(b), C₃₋₈cycloalkyl, C₃₋₈cycloalkylY,C(═O)NR^(a)R^(b), CF₃, OCF₃, CN, SO₂NR^(a)R^(b),OC₂₋₄alkyleneNR^(a)R^(b), and C(═O)NR^(a)R^(c), or R⁴ and R⁵ are takentogether with the carbon atoms to which they are attached to form a 5-or 6-membered ring, saturated or partially or fully saturated,optionally substituted and optionally containing one or two heteroatoms.

An especially preferred subclass of compounds within the general scopeof formula (I) is represented by compounds of formula (II)

and pharmaceutically acceptable salts and solvates (e.g., hydrates)thereof.

Compounds of formula (I) can contain one or more asymmetric center, and,therefore, can exist as stereoisomers. The present invention includesboth mixtures and separate individual stereoisomers of the compounds offormula (I). Compounds of formula (I) also can exist in tautomericforms, and the invention includes both mixtures and separate individualtautomers thereof.

Pharmaceutically acceptable salts of the compounds of formula (I) can beacid addition salts formed with pharmaceutically acceptable acids.Examples of suitable salts include, but are not limited to, thehydrochloride, hydrobromide, sulfate, bisulfate, phosphate, hydrogenphosphate, acetate, benzoate, succinate, fumarate, maleate, lactate,citrate, tartrate, gluconate, methanesulfonate, benzenesulfonate, andp-toluenesulfonate salts. The compounds of the formula (I) also canprovide pharmaceutically acceptable metal salts, in particular alkalimetal salts and alkaline earth metal salts, with bases. Examples includethe sodium, potassium, magnesium, and calcium salts.

Compounds of the present invention are potent and selective inhibitorsof cGMP-specific PDE5. Thus, compounds of formula (I) are of interestfor use in therapy, specifically for the treatment of a variety ofconditions where selective inhibition of PDE5 is considered to bebeneficial.

Phosphodiesterases (PDEs) catalyze the hydrolysis of cyclic nucleotides,such as cyclic adenosine monophosphate (cAMP) and cyclic guanosinemonophosphate (cGMP). The PDEs have been classified into at least sevenisoenzyme families and are present in many tissues (J. A. Beavo,Physiol. Rev., 75, p. 725 (1995)).

PDE5 inhibition is a particularly attractive target. A potent andselective inhibitor of PDE5 provides vasodilating, relaxing, anddiuretic effects, all of which are beneficial in the treatment ofvarious disease states. Research in this area has led to several classesof inhibitors based on the cGMP basic structure (E. Sybertz et al.,Expert. Opin. Ther. Pat., 7, p. 631 (1997)).

The biochemical, physiological, and clinical effects of PDE5 inhibitorstherefore suggest their utility in a variety of disease states in whichmodulation of smooth muscle, renal, hemostatic, inflammatory, and/orendocrine function is desirable. The compounds of formula (I),therefore, have utility in the treatment of a number of disorders,including stable, unstable, and variant (Prinzmetal) angina,hypertension, pulmonary hypertension, congestive heart failure, acuterespiratory distress syndrome, acute and chronic renal failure,atherosclerosis, conditions of reduced blood vessel patency (e.g.,postpercutaneous transluminal coronary or carotid angioplasty, orpost-bypass surgery graft stenosis), peripheral vascular disease,vascular disorders, such as Raynaud's disease, thrombocythemia,inflammatory diseases, stroke, bronchitis, chronic asthma, allergicasthma, allergic rhinitis, glaucoma, osteoporosis, preterm labor, benignprostatic hypertrophy, peptic ulcer, male erectile dysfunction, femalesexual dysfunction, and diseases characterized by disorders of gutmotility (e.g., irritable bowel syndrome).

An especially important use is the treatment of male erectiledysfunction, which is one form of impotence and is a common medicalproblem. Impotence can be defined as a lack of power, in the male, tocopulate, and can involve an inability to achieve penile erection orejaculation, or both. The incidence of erectile dysfunction increaseswith age, with about 50% of men over the age of 40 suffering from somedegree of erectile dysfunction.

In addition, a further important use is the treatment of female arousaldisorder, also termed female sexual arousal disorder. Female arousaldisorders are defined as a recurrent inability to attain or maintain anadequate lubrication/swelling response of sexual excitement untilcompletion of sexual activity. The arousal response consists ofvasocongestion in the pelvis, vaginal lubrication, and expansion andswelling of external genitalia.

It is envisioned, therefore, that compounds of formula (I) are useful inthe treatment of male erectile dysfunction and female sexual arousaldisorder. Thus, the present invention concerns the use of compounds offormula (I), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing either entity, for the manufactureof a medicament for the curative or prophylactic treatment of erectiledysfunction in a male animal and sexual arousal disorder in a femaleanimal, including humans.

The term “treatment” includes preventing, lowering, stopping, orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” includes both medical therapeuticand/or prophylactic administration, as appropriate.

It also is understood that “a compound of formula (I),” or aphysiologically acceptable salt or solvate thereof, can be administeredas the neat compound, or as a pharmaceutical composition containingeither entity.

Although the compounds of the invention are envisioned primarily for thetreatment of sexual dysfunction in humans, such as male erectiledysfunction and female sexual arousal disorder, they also can be usedfor the treatment of other disease states. A further aspect of thepresent invention, therefore, is providing a compound of formula (I) foruse in the treatment of stable, unstable, and variant (Prinzmetal)angina, hypertension, pulmonary hypertension, chronic obstructivepulmonary disease, congestive heart failure, acute respiratory distresssyndrome, acute and chronic renal failure, atherosclerosis, conditionsof reduced blood vessel patency (e.g., post-PTCA or post-bypass graftstenosis), peripheral vascular disease, vascular disorders such asRaynaud's disease, thrombocythemia, inflammatory diseases, prophylaxisof myocardial infarction, prophylaxis of stroke, stroke, bronchitis,chronic asthma, allergic asthma, allergic rhinitis, glaucoma,osteoporosis, preterm labor, benign prostatic hypertrophy, male andfemale erectile dysfunction, or diseases characterized by disorders ofgut motility (e.g., IBS).

According to another aspect of the present invention, there is providedthe use of a compound of formula (I) for the manufacture of a medicamentfor the treatment of the above-noted conditions and disorders.

In a further aspect, the present invention provides a method of treatingthe above-noted conditions and disorders in a human or nonhuman animalbody which comprises administering to said body a therapeuticallyeffective amount of a compound of formula (I).

Compounds of the invention can be administered by any suitable route,for example by oral, buccal, inhalation, sublingual, rectal, vaginal,transurethral, nasal, topical, percutaneous, i.e., transdermal, orparenteral (including intravenous, intramuscular, subcutaneous, andintracoronary) administration. Parenteral administration can beaccomplished using a needle and syringe, or using a high pressuretechnique, like POWDERJECT™.

Oral administration of a compound of the invention is the preferredroute. Oral administration is the most convenient and avoids thedisadvantages associated with other routes of administration. Forpatients suffering from a swallowing disorder or from impairment of drugabsorption after oral administration, the drug can be administeredparenterally, e.g., sublingually or buccally.

Compounds and pharmaceutical compositions suitable for use in thepresent invention include those wherein the active ingredient isadministered in an effective amount to achieve its intended purpose.More specifically, a “therapeutically effective amount” means an amounteffective to prevent development of, or to alleviate the existingsymptoms of, the subject being treated. Determination of the effectiveamounts is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

A “therapeutically effective dose” refers to that amount of the compoundthat results in achieving the desired effect. Toxicity and therapeuticefficacy of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, which is expressed as the ratio between LD₅₀ and ED₅₀. Compoundswhich exhibit high therapeutic indices are preferred. The data obtainedfrom such data can be used in formulating a range of dosage for use inhumans. The dosage of such compounds preferably lies within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed, and the route of administration utilized.

The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition. Dosageamount and intervals can be adjusted individually to provide plasmalevels of the active moiety which are sufficient to maintain therapeuticeffects.

The amount of composition administered is dependent on the subject beingtreated, the subject's weight, the severity of the affliction, themanner of administration, and the judgment of the prescribing physician.

Specifically, for administration to a human in the curative orprophylactic treatment of the conditions and disorders identified above,oral dosages of a compound of formula (I) generally are about 0.5 toabout 1000 mg daily for an average adult patient (70 kg). Thus, for atypical adult patient, individual tablets or capsules contain 0.2 to 500mg of active compound, in a suitable pharmaceutically acceptable vehicleor carrier, for administration in single or multiple doses, once orseveral times per day. Dosages for intravenous, buccal, or sublingualadministration typically are 0.1 to 500 mg per single dose as required.In practice, the physician determines the actual dosing regimen which ismost suitable for an individual patient, and the dosage varies with theage, weight, and response of the particular patient. The above dosagesare exemplary of the average case, but there can be individual instanceswherein higher or lower dosages are merited, and such are within thescope of this invention.

For human use, a compound of the formula (I) can be administered alone,but generally is administered in admixture with a pharmaceutical carrierselected with regard to the intended route of administration andstandard pharmaceutical practice. Pharmaceutical compositions for use inaccordance with the present invention thus can be formulated in aconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries that facilitateprocessing of compounds of formula (I) into preparations which can beused pharmaceutically.

These pharmaceutical compositions can be manufactured in a conventionalmanner, e.g., by conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Proper formulation is dependent upon the routeof administration chosen. When a therapeutically effective amount of acompound of the present invention is administered orally, thecomposition typically is in the form of a tablet, capsule, powder,solution, or elixir. When administered in tablet form, the compositioncan additionally contain a solid carrier, such as a gelatin or anadjuvant. The tablet, capsule, and powder contain about 5% to about 95%compound of the present invention, and preferably from about 25% toabout 90% compound of the present invention. When administered in liquidform, a liquid carrier such as water, petroleum, or oils of animal orplant origin can be added. The liquid form of the composition canfurther contain physiological saline solution, dextrose or othersaccharide solutions, or glycols. When administered in liquid form, thecomposition contains about 0.5% to about 90% by weight of a compound ofthe present invention, and preferably about 1% to about 50% of acompound of the present invention.

When a therapeutically effective amount of a compound of the presentinvention is administered by intravenous, cutaneous, or subcutaneousinjection, the composition is in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable solutions, having due regard to pH, isotonicity,stability, and the like, is within the skill in the art. A preferredcomposition for intravenous, cutaneous, or subcutaneous injectiontypically contains, in addition to a compound of the present invention,an isotonic vehicle.

For oral administration, the compounds readily can be formulated bycombining a compound of formula (I) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the presentcompounds to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by adding a compound of formula (I) with asolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients include, forexample, fillers and cellulose preparations. If desired, disintegratingagents can be added.

For administration by inhalation, compounds of the present invention areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant.In the case of a pressurized aerosol, the dosage unit can be determinedby providing a valve to deliver a metered amount. Capsules andcartridges of, e.g., gelatin, for use in an inhaler or insufflator canbe formulated containing a powder mix of the compound and a suitablepowder base such as lactose or starch.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multidose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing, and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils or synthetic fatty acid esters. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension. Optionally, the suspension also can containsuitable stabilizers or agents that increase the solubility of thecompounds and allow for the preparation of highly concentratedsolutions. Alternatively, a present composition can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

Compounds of the present invention also can be formulated in rectalcompositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases. In addition to theformulations described previously, the compounds also can be formulatedas a depot preparation. Such long-acting formulations can beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Many of the compounds of the present invention can be provided as saltswith pharmaceutically compatible counterions. Such pharmaceuticallyacceptable base addition salts are those salts that retain thebiological effectiveness and properties of the free acids, and that areobtained by reaction with suitable inorganic or organic bases.

In particular, a compound of formula (I) can be administered orally,buccally, or sublingually in the form of tablets containing excipients,such as starch or lactose, or in capsules or ovules, either alone or inadmixture with excipients, or in the form of elixirs or suspensionscontaining flavoring or coloring agents. Such liquid preparations can beprepared with pharmaceutically acceptable additives, such as suspendingagents. A compound also can be injected-parenterally, for example,intravenously, intramuscularly, subcutaneously, or intracoronarily. Forparenteral administration, the compound is best used in the form of asterile aqueous solution which can contain other substances, forexample, salts, or monosaccharides, such as mannitol or glucose, to makethe solution isotonic with blood.

For veterinary use, a compound of formula (I) or a nontoxic saltthereof, is administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal.

Thus, the invention provides in a further aspect a pharmaceuticalcomposition comprising a compound of the formula (I), together with apharmaceutically acceptable diluent or carrier therefor. There isfurther provided by the present invention a process of preparing apharmaceutical composition comprising a compound of formula (I), whichprocess comprises mixing a compound of formula (I), together with apharmaceutically acceptable diluent or carrier therefor.

In a particular embodiment, the invention includes a pharmaceuticalcomposition for the curative or prophylactic treatment of erectiledysfunction in a male animal, or arousal disorder in a female animal,including humans, comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable diluent or carrier.

Compounds of formula (I) can be prepared by any suitable method known inthe art, or by the following processes which form part of the presentinvention. In the methods below, R¹, R², R³, R⁴, and R⁵ are as definedin structural formula (I) above. In particular, Daugan U.S. Pat. No.5,859,006, incorporated herein by reference, discloses preparation of acompound of structural formula (III).

Daugan U.S. Pat. No. 5,859,006 teaches the preparation of the compoundof structural formula (III), wherein R⁰ is H, beginning with atryptophan ester having the structural formula (IV):

The compounds of structural formula (I) can be prepared in an analogousmanner as a compound of structural formula (III) using an appropriatelysubstituted tryptophan ester of structural formula (V):

Many substituted tryptophan esters of structural formula (V) arecommercially available, and, if necessary, can be converted to othersubstituted esters. Substituted tryptophan esters also can be prepared,for example, from substituted indoles, as set forth in S. Wagaw et al.,J. Amer. Chem. Soc., 21, p. 10251 (1999) and M. P. Moyer et al., J. Org.Chem., 51, p. 5106 (1986). These substituted tryptophan esters can beused in the synthetic methods disclosed in Daugan U.S. Pat. No.5,859,006 to provide compositions of structural formula (I).

It should be understood that protecting groups can be utilized inaccordance with general principles of organic synthetic chemistry toprovide compounds of structural formula (I). Protecting compounds andprotecting groups, like benzyl chloroformate and trichloroethylchloroformate, are well known to persons skilled in the art, forexample, see T. W. Greene et al., “Protective Groups in OrganicSynthesis, Third Edition,” John Wiley and Sons, Inc., NY, N.Y. (1999).These protecting groups are removed when necessary by appropriate basic,acidic, or hydrogenolytic conditions known to persons skilled in theart. Accordingly, compounds of structural formula (I) not specificallyexemplified herein can be prepared by persons skilled in the art.

In addition, compounds of formula (I) can be converted to othercompounds of formula (I). Thus, for example, a particular R⁴ substituentcan be interconverted to prepare another suitably substituted compoundof formula (I). Examples of appropriate interconversions include, butare not limited to, nitro to amino, OR^(a) to hydroxy by suitable means(e.g., using a reducing agent such as SnCl₂ or a palladium catalyst,such as palladium-on-carbon), or amino to substituted amino, such asacylamino or sulphonylamino, using standard acylating or sulfonylatingconditions. In cases wherein R¹ represents a substituted bicyclicsystem, suitable interconversion can involve removal of a substituent,such as by treatment with a palladium catalyst whereby, for example, abenzyl substituent is removed from a suitable bicyclic system.

Compounds of formula (I) can be prepared by the method above asindividual stereoisomers from the appropriate stereoisomer of formula(IV) or as a racemic mixture from the appropriate racemic compound offormula (IV). Individual stereoisomers of the compounds of the inventioncan be prepared from racemates by resolution using methods known in theart for the separation of racemic mixtures into their constituentstereoisomers, for example, using HPLC on a chiral column, such asHypersil naphthyl urea, or using separation of salts of stereoisomers.Compounds of the invention can be isolated in association with solventmolecules by crystallization from, or evaporation of, an appropriatesolvent.

The pharmaceutically acceptable acid addition salts of the compounds offormula (I) that contain a basic center can be prepared in aconventional manner. For example, a solution of the free base can betreated with a suitable acid, either neat or in a suitable solution, andthe resulting salt isolated either by filtration or by evaporation undervacuum of the reaction solvent. Pharmaceutically acceptable baseaddition salts can be obtained in an analogous manner by treating asolution of a compound of formula (I) with a suitable base. Both typesof salt can be formed or interconverted using ion-exchange resintechniques. Thus, according to a further aspect of the invention, amethod for preparing a compound of formula (I) or a salt or solvate(e.g., hydrate) is provided, followed by (i) salt formation, or (ii)solvate (e.g., hydrate) formation.

The following abbreviations are used hereafter in the accompanyingexamples: rt (room temperature), min (minute), h (hour), g (gram), mmol(millimole), m.p. (melting point), eq (equivalents), aq (aqueous), L(liter), mL (milliliter), μL (microliter), DMSO (dimethyl sulfoxide),CH₂Cl₂ (dichloromethane), IPA (isopropyl alcohol), TFA (trifluoroaceticacid), NaOH (sodium hydroxide), TsOH (p-toluenesulfonic acid), Me(methyl), Et (ethyl), EtOH (ethanol), MeOH (methanol), DMF(dimethylformamide), Et₃N (triethylamine), MeNH₂ (methylamine), HOAC(acetic acid), Ac₂O (acetic anhydride), Ac (C(═O)CH₃), and THF(tetrahydrofuran).

PREPARATION OF EXAMPLE 1

EXAMPLE 1

Example 1 was prepared from 5-hydroxy-DL-tryptophan as depicted in thefollowing synthetic scheme. 5-Hydroxy-DL-tryptophan is a commerciallyavailable compound from Aldrich Chemical Co., Milwaukee, Wis.

Intermediate 1Preparation of 5-hydroxy-DL-tryptophan methyl ester hydrochloride

Thionyl chloride (2.14 mL, 29.4 mmol) was added dropwise to a slurry of5-hydroxy-DL-tryptophan (3.8 g, 17.3 mmol) in anhydrous methanol (40 mL)at 0° C. under a nitrogen blanket. The resulting mixture was warmedslowly to room temperature and stirred for 17 hours. Then the solventwas removed under reduced pressure to provide 5-hydroxy-DL-tryptophanmethyl ester hydrochloride (Intermediate 1) as a light brown foam (5.0g, 100%): ¹H NMR (300 MHz, DMSO-d₆): δ 10.78 (bs, 1H), 8.62-8.25 (m,3H), 7.18-7.11 (m, 2H), 6.79 (s, 1H), 6.67-6.61 (m, 1H), 4.18 (m, 1H),3.69 (s, 3H), 3.25-3.12 (m, 2H).

Intermediate 2

Preparation of a cis-β-Carboline

A mixture of Intermediate 1 (2.7 g, 10 mmol) and piperonal (1.8 g, 12mmol) in IPA (27 mL) was heated at reflux under a nitrogen blanket for 8hours, during which time a precipitate formed (after about 4 hours). Theresulting slurry was cooled to room temperature, vacuum filtered, andthe solid was washed with IPA (2×5 mL). The filtrate solvent was removedunder reduced pressure, then the residue was diluted with ethyl acetate(60 mL) and neutralized with saturated sodium bicarbonate (NaHCO₃)solution (20 mL). The basic aqueous layer was extracted with ethylacetate, then the combined organic extracts were concentrated underreduced pressure to provide a yellow solid residue. The residue waspurified by column chromatography, eluting with methylene chloride/ethylacetate (4:1), to provide Intermediate 2 as a yellow solid (420 mg,12%): TLC R_(f) (4:1 methylene chloride/ethyl acetate)=0.55; ¹H NMR (300MHz, CDCl₃): δ 7.31 (bs, 1H), 7.07 (d, J=8.5 Hz, 1H), 6.92-6.68 (m, 5H),5.95 (s, 2H), 5.14 (s, 1H), 3.97-3.91 (m, 1H), 3.81 (s, 3H), 3.19-3.08(m, 1H), 3.00-2.88 (m 1H). The trans carboline also was obtained as ayellow solid, but was not characterized (420 mg, 12%): TLC R_(f) (4:1methylene chloride/ethyl acetate)=0.20.

Intermediate 3

Preparation of (+/−)-cis-2-chloroacetyl-β-carboline

Chloroacetyl chloride (0.36 mL, 4.52 mmol) was added dropwise to amixture of Intermediate 2 (500 mg, 1.28 mmol) and triethylamine (0.63mL, 4.54 mmol) in methylene chloride (20 μL) at 0° C. under a nitrogenblanket, and the resulting mixture was stirred at 5° C. for 2.5 hours.The resulting brown solution was diluted with methylene chloride (40 mL)and washed successively with saturated NaHCO₃ (15 mL) and brine (10 mL).The organic layer was dried over sodium sulfate (Na₂SO₄), then thesolvent was removed under reduced pressure to provide Intermediate 3 asa thick yellow oil (0.95 g), which was used without purification: TLCR_(f) (4:1 methylene chloride/ethyl acetate)=0.92.

EXAMPLE 1

Preparation of (+/−,cis)-6-Benzo[1,3]dioxol-5-yl-10-hydroxy-2-methyl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6)pyrido[3,4-b]indole-1,4-dione

A mixture of crude Intermediate 3 (0.95 g), methylamine (4.5 mL, 2.0M inTHF, 9 mmol), and methanol (8 mL) was heated at 50° C. under a nitrogenblanket for 5 hours, after which the resulting mixture was heated for 17hours at 40° C. The resulting orange slurry was cooled to roomtemperature, then the precipitate was collected by vacuum filtration.The solid was washed with methanol (5×2 mL), slurried in diethyl etherfor 5 hours, then the solid was collected by vacuum filtration. Thesolid next was dried in a vacuum oven at 70° C. for 24 hours to providethe compound of Example 1 as a white powder (0.33 g, 64% for two steps).The desired cis isomer was confirmed by an NOE difference experiment(1.0% enhancement): mp 322-328° C.; TLC R_(f) (4:1:0.4 methylenechloride/ethyl acetate/methanol)=0.55; ¹H NMR (300 MHz, DMSO-d₆): δ10.70 (s, 1H), 8.67 (s, 1H), 7.08 (d, J=8.6 Hz, 1H), 6.85-6.75 (m, 3H),6.60-6.55 (m, 1H), 6.09 (s, 1H), 5.93 (s, 2H), 4.42-4.33 (m, 1H), 4.18(d, J=17.0 Hz, 1H), 3.93 (d, J=17.0 Hz, 1H), 3.45-3.32 (m, 1H),3.00-2.88 (m, 4H); ¹³C NMR (75 MHz, DMSO-d₆): δ 166.8, 166.6, 150.6,146.9, 145.9, 137.0, 134.2, 130.6, 136.4, 119.1, 111.6, 111.2, 107.9,106.8, 103.9, 102.1, 100.8, 55.4, 55.1, 51.4, 32.8, 23.0 ppm; CI MS(methane) m/z 406 [M+H]⁺. Anal. Calcd. for C₂₂H₁₉N₃O₅; C, 65.18; H,4.72; N, 10.37. Found: C, 64.47; H, 5.04; H, 10.24.

PREPARATION OF EXAMPLE 2

Example 2 was prepared from 5-methoxy-DL-tryptophan as depicted in thefollowing synthetic scheme. 5-Methoxy-DL-tryptophan is a commerciallyavailable compound from Aldrich Chemical Co., Milwaukee, Wis.

Intermediate 4Preparation of 5-Methoxy-DL-tryptophan methyl ester

Thionyl chloride (1.0 mL, 13.7 mmol) was added dropwise to a slurry of5-methoxy-DL-tryptophan (2.0 g, 8.54 mmol) in anhydrous methanol (22 mL)at 0° C. under a nitrogen blanket. The resulting solution was warmedslowly to room temperature, stirred for 17 hours, then the solvent wasremoved under reduced pressure. The resulting residue was dissolved inwater (20 mL), neutralized with saturated NaHCO₃ (10 mL), and extractedtwice with methylene chloride. The combined organic layers were driedover Na₂SO₄, filtered, and the solvent was removed under reducedpressure to provide 5-methoxy-DL-tryptophan methyl ester (Intermediate4) as a light brown solid (1.55 g, 73%): ¹H NMR (300 MHz, CDCl₃): δ 7.93(bs, 1H), 7.30-7.25 (m, 1H), 7.09-7.03 (m, 1H), 6.90-6.83 (m, 1H),3.90-3.78 (m, 4H), 3.72 (s, 3H), 3.30-3.20 (m, 1H), 3.10-2.98 (m, 1H).

Intermediate 5

Preparation of the (+/−)-cis-β-Carboline

Trifluoroacetic acid (0.65 mL, 8.48 mmol) was added to a mixture ofIntermediate 4 (1.6 g, 6.45 mmol) and piperonal (1.17 g, 7.74 mmol) inmethylene chloride (30 mL) at 0° C. under a nitrogen blanket. Theresulting mixture was warmed to room temperature then stirred for 48hours. The reaction mixture was diluted with methylene chloride (70 mL),then neutralized with saturated NaHCO₃ (15 mL). The organic layer wasconcentrated under reduced pressure, and the residue was purified byflash column chromatography, eluting with methylene chloride/ethylacetate (8:1), to provide Intermediate 5 as a yellow solid (882 mg.36%): TLC R_(f) (4:1 methylene chloride/ethyl acetate)=0.69; ¹H NMR (300MHz, CDCl₃): δ 7.32 (bs, 1H), 7.14 (d, J=8.7 Hz, 1H), 7.00 (S, 1H),6.93-6.80 (m, 4H), 5.97 (S, 2H), 5.18 (s, 1H), 4.02-3.94 (m, 1H), 3.88(s, 3H), 3.84 (s, 3H), 3.27-3.16 (m, 1H), 3.07-2.96 (m, 1H). The transcarboline also was obtained as a yellow solid, but was not characterized(850 mg, 35%): TLC R_(f) (4:1: methylene chloride/ethyl acetate)=0.40.

Intermediate 6

Preparation of the (+/−)-cis-2-Chloroacetyl-β-carboline

Chloroacetyl chloride (0.21 mL, 2.60 mmol) was added dropwise to amixture of Intermediate 5 (880 mg, 2.17 mmol) and triethylamine (0.36mL, 2.60 mmol) in methylene chloride (18 mL) at 0° C. under a nitrogenblanket, and the resulting mixture was stirred at 0° C. for 2 hours. Theresulting solution then was diluted with methylene chloride (60 mL),washed with saturated NaHCO₃ (10 mL), dried over sodium sulfate,filtered, and the solvent was removed under reduced pressure to provideIntermediate 6 as a yellow foam which was used without purification(1.14 g): TLC R_(f) (4:1 methylene chloride/ethyl acetate)=0.92.

EXAMPLE 2

Preparation of(trans)-6-(1,3-Benzodioxol-5-yl)-10-methoxy-2-methyl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione

A mixture of crude Intermediate 6 (1.14 g, 2.17 mmol) and methylamine (5mL, 2.0 M in THF, 10 mmol) in methanol (12 mL) was heated at 50° C.under a nitrogen blanket for 6 hours, after which the mixture wasstirred at room temperature for 17 hours. The resulting precipitate wasisolated by vacuum filtration, then the solid was washed with methanol(2×5 mL) and dried in a vacuum oven at 60° C. for 24 hours to provideExample 2 as a white powder (0.65 g, 71% over two steps): mp 259-261°C.; TLC R_(f) (4:1 methylene chloride/ethyl acetate)=0.35; ¹H NMR (300MHz, DMSO-d₆): δ 10.81 (s, 1H), 7.18 (d, J=8.8 Hz, 1H), 7.09 (d, J=1.5Hz, 1H), 6.85 (s, 1H), 6.78 (s, 2H), 6.72-6.62 (m, 1H), 6.10 (s, 1H),5.93 (s, 2H), 4.43-4.32 (m, 1H), 4.18 (d, J=17.2 Hz, 1H), 3.94 (d,J=17.2 Hz, 1H), 3.57-3.45 (m 1H), 3.01-2.82 (m, 4H): ¹³C NMR (75 MHz,DMSO-d₆) δ: 166.8, 166.6, 153.4, 147.0, 146.0, 137.0, 134.5, 131.1,126.1, 119.2, 111.9, 111.2, 107.9, 106.9, 104.6, 100.8, 100.2, 55.5,55.3, 55.2, 51.4, 32.8, 23.2 ppm; CI MS (methane) m/z 420[C₂₃H₂₁N₃O₅+H]⁺. Anal. Calcd. for C₂₃H₂₁N₃O₅: C, 65.86; H, 5.05; N,10.02. Found: C, 65.75; H, 4.95; N, 9.91. The relative stereochemistryof Example 2 was confirmed to be cis isomer by an NOE differenceexperiment (DMSO-d₆): a positive NOE enhancement (2.3%) from the C12aproton at 4.37 ppm to the C6 proton at 6.12 ppm.

PREPARATION OF EXAMPLE 3

Example 3 was prepared from 6-methoxy-D-tryptophan ethyl ester asdepicted in the following synthetic scheme. 6-Methoxy-D-tryptophan ethylester is a commercially available compound from Aldrich Chemical Col,Milwaukee, Wis.

Intermediate 7Preparation of a cis-β-Carboline

Trifluoroacetic acid (0.295 mL, 3.82 mmol) was added to a mixture of6-methoxy-D-tryptophan ethyl ester (500 mg, 1.91 mmol) and piperonal(573 mg, 3.82 mmol) in methylene chloride (15 mL) at 0° C. under anitrogen blanket, after which the mixture was warmed to room temperatureovernight, then stirred for 3 days. The reaction mixture then wasdiluted with methylene chloride (30 mL) and neutralized with saturatedNaHCO₃ (5 mL). The organic layer then was washed with brine (5 mL),dried over Na₂SO₄, filtered, and the solvent was removed under reducedpressure to provide a brown solid residue. The residue was purified byflash column chromatography, eluting with methylene chloride/ethylacetate (10:1), to provide Intermediate 7 as a yellow solid (120 mg,15%): TLC R_(f) (10:1 methylene chloride/ethyl acetate)=0.57; ¹H NMR(300 MHz, CDCl₃): δ 7.42-7.30 (m, 2H), 6.90-6.70 (m, 6H), 5.94 (s, 2H),5.13 (s, 1H), 4.31-4.19 (m, 2H), 3.93-3.86 (m, 1H), 3.79 (s, 3H),3.20-3.10 (m, 1H), 3.00-2.89 (m, 1H), 1.35 (t, J=8.5 Hz, 3H). Thetrans-β-carboline also was obtained as a yellow solid, but was notcharacterized (160 mg, 20%): TLC R_(f) (10:1 methylene chloride/ethylacetate)=0.29.

Intermediate 8

Preparation of a cis-2-Chloroacetyl-β-carboline

Chloroacetyl chloride (0.030 mL, 0.373 mmol) was added dropwise to amixture of Intermediate 7 (120 mg, 0.28.7 mmol) and triethylamine (0.052mL, 0.373 mmol) in methylene chloride (5 mL) at 0° C. under a nitrogenblanket, and the resulting mixture was stirred at 0° C. for 1.5 hours.The brown solution then was diluted with methylene chloride (30 mL),washed with saturated NaHCO₃ (5 mL) and brine (5 mL), and the solventwas removed under reduced pressure to provide Intermediate 8 as a yellowsolid which was used without further purification (179 mg): TLC R_(f)(4:1 methylene chloride/ethyl acetate)=0.92.

EXAMPLE 3

Preparation of(6R,12aR)-6-Benzo[1,3]dioxol-5-yl-9-methoxy-2-methyl-2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione

A mixture of crude Intermediate 8 (179 mg) and methylamine (0.5 mL, 2.0Min THF, 1.0 mmol) in THF (3 mL) and methanol (5 mL) was heated at 50° C.under a nitrogen blanket for 6 hours. A second portion of methylamine(0.25 mL, 0.5 mmol) was added, and the resulting mixture was stirred at40° C. for an additional 16 hours. The resulting orange slurry wascooled to room temperature, then the mixture was concentrated to about 5mL. The resulting precipitate was isolated by vacuum filtration. Thesolid then was washed with methanol (3×2 mL) and dried in a vacuum ovenat 70° C. to provide Example 3 as an off-white powder (0.085 g, 71% overtwo steps): mp 288-293° C.; TLC R_(f) (4:1 methylene chloride/ethylacetate)=0.38; ¹H NMR (300 MHz, DMSO-d₆): δ 10.81 (s, 1H), 7.42 (d,J=8.6 Hz, 1H), 6.84 (s, 1H), 6.80 (s, 1H), 6.77 (s, 2H), 6.65 (dd,J=8.6, 2.1 Hz, 1H), 6.09 (s, 1H), 5.92 (s, 2H), 4.38 (dd, J=11.6, 4.3Hz, 1H), 4.17 (d, J=19.4 Hz, 1H), 3.93 (d, J=19.4 Hz, 1H), 3.73 (s, 3H),3.46 (dd, J=15.9, 4.5 Hz, 1H), 3.00-2.87 (m, 4H); API m/z 420[C₂₃H₂₁N₃O₅+H]⁺; [α]_(D) ^(25° C.)=+136.7° (c=0.25, DMSO). Anal. Calcd.for C₂₃H₂₁N₃O₅; C, 65.86; H, 5.05; N, 10.02. Found: C, 65.96; H, 4.97;N, 9.91. The stereochemistry of Example 3 was confirmed to be thedesired cis isomer by a series of NOE difference experiments: a positiveNOE enhancement from the C12a proton at 4.38 ppm to the C6 proton at6.09 ppm; a positive NOE enhancement from the C6 proton at 6.09 ppm tothe C12a proton at 4.38 ppm. Chiral HPLC analysis (Chiralcel OD Column,250×4.6 mm, Retention Time=20.9 minutes; 1:1 isopropanol/hexanes;flow=1.0 mL/minute; detector@220 nm; 25° C.) showed one major peak, witha purity of 99.8%.

PREPARATION OF EXAMPLE 4

Example 4 was prepared from 6-bromo-DL-tryptophan ethyl esterhydrochloride, i.e., Intermediate 9, as depicted in the followingsynthetic scheme. Intermediate 9 was prepared from4-bromo-2-nitrotoluene in four well-known synthetic steps.4-Bromo-2-nitrotoluene is a commercially available compound from AldrichChemical Co., Milwaukee, Wis.

Intermediate 11Preparation of 6-Bromoindole

The synthesis of Intermediate 11 is disclosed in M. P. Moyer et al., J.Org. Chem., 51, page 5106 (1986); and K. L. Rinehart et al., J. Am.Chem. Soc., 109, page 3378 (1987).

A mixture of 4-bromo-2-nitrotoluene (20 g, 93 mmol), pyrrolidine (8 mL,93 mmol), and N,N-dimethylformamide dimethyl acetal (37 mL, 278 mmol) inanhydrous N,N-dimethylformamide (200 mL) was heated at 110° C. under anitrogen blanket for 2 hours. The mixture was cooled, then diluted withwater (1.5 L) and extracted with ether (3×500 mL). The combined organicextracts were washed with brine (500 mL), dried over Na₂SO₄, andfiltered. The solvent was removed under reduced pressure to provideIntermediate 10 as dark purple crystals which were suitable for usewithout further purification (27.9 g): TLC R_(f) (1:4 ethylacetate/chloroform)=0.77.

Zinc granules (30 g, 459 mmol) were added over 30 minutes to a solutionof crude Intermediate 10 (25 g, 93 mmol) in acetic acid (400 mL) andwater (100 mL) at 75° C., after which the mixture was stirred for anadditional 1 hour. The suspension was cooled, filtered, and extractedwith ethyl acetate (2×2 L). The combined organic extracts were washedwith water (2×2 L), saturated NaHCO₃ solution (2×500 mL), and brine(2×500 mL), dried over magnesium sulfate (MgSO₄), and filtered. Thesolvent was removed under reduced pressure to yield a purple residuewhich was purified by flash column chromatography, eluting withhexanes/methylene chloride (1:1), to provide 6-bromoindole (Intermediate11) as a blue-white powder (4.9 g, 27% over two steps): TLC R_(f) (1:9ethyl acetate/chloroform)=0.81. The proton NMR spectrum (300 MHz, CDCl₃)was identical to the known compound.

Intermediate 9

Preparation of 6-Bromo-DL-tryptophan ethyl ester hydrochloride

The synthesis of Intermediate 12 is disclosed in T. L. Gilchrist et al.,J. Chem. Soc., Chem. Commun., page 1089 (1979); U. Schmidt et al.,Liebigs Ann. Chem., page 785 (1985); and U. Schmidt et al., TetrahedronLett., 23, page 4911 (1982).

Ethyl bromopyruvate (10 mL, 80 mmol) was added dropwise to a biphasicmixture of hydroxyl-amine sulfate (6.6 g, 40 mmol) in water (50 mL) andchloroform (50 mL), and the mixture was stirred at room temperature for3 hours. The mixture was filtered, and the organic phase wasconcentrated under reduced pressure to yield the oxime Intermediate 15as a white solid, which was used without further purification (15.9 g,95%): TLC R_(f) (1:4 ethyl acetate/chloroform)=0.12.

Powdered sodium carbonate (4.0 g, 37 mmol) was added to a dark greensolution of Intermediate 11 (4.9 g, 25 mmol) and Intermediate 15 (7.9 g,37 mmol) in methylene chloride (50 mL) at room temperature under anitrogen blanket. The mixture was stirred for 7 hours, after which themixture was diluted with ethyl acetate (150 mL), washed with brine (100mL), dried over MgSO₄, and filtered. The solvents were removed underreduced pressure to provide Intermediate 12 as a dark green oil whichwas suitable for use without further purification (9.6 g): TLC R_(f)(1:9 ethyl acetate/chloroform)=0.18.

Aluminum amalgam was prepared by sequentially dipping several pieces ofaluminum foil (5 g total) in 1 N sodium hydroxide, saturated mercury(II) chloride solution, water, and ethanol. The aluminum pieces thenwere added to a solution of crude Intermediate 12 in ether (100 mL) andwater (5 mL) at room temperature. The gray suspension was stirred for 24hours, then filtered. The organic layer was washed with brine (100 mL),dried over Na₂SO₄, and filtered. The solvents were removed underpressure to provide a dark green residue, which was treated with aethereal HCl (1 M in ether). The solid was collected by vacuumfiltration, and dried in a vacuum oven at 60° C. overnight to provideIntermediate 9 as a darkred powder (3.3 g, 38% over two steps): ¹H NMR(300 MHz, DMSO-d₆): δ 11.21 (s, 1H), 8.40 (s, 2H), 7.57 (s, 1H), 7.48(d, J=8.4 Hz, 1H), 7.27 (s, 1H), 7.16 (d, J=7.0 Hz, 1H), 4.24-4.18 (m,3H), 4.09 (q, J=6.8 Hz, 2H), 1.10 (t, J=7.3 Hz, 3H).

Intermediate 13

Preparation of a cis-β-carboline

A solution of saturated aqueous sodium carbonate (50 mL) was added to adark green solution of Intermediate 9 (3.4 g, 10 mmol) in methylenechloride (100 mL), and the resulting mixture was shaken for 10 minutes.The organic layer then was dried over Na₂SO₄ and filtered. The solutionwas concentrated under reduced pressure to a volume of 20 mL and cooledto 0° C. under a nitrogen blanket. Piperonal (1.7 g, 12 mmol) was addedto the solution, followed by the addition of trifluoroacetic acid (1.5mL, 20 mmol), after which the solution was warmed to room temperatureovernight. The reaction mixture then was diluted with ethyl acetate, andneutralized with aqueous NaHCO₃ (100 mL). The organic layer was washedwith brine (50 mL), dried over Na₂SO₄, filtered, and the solvent wasremoved under reduced pressure to provide a brown oil. This residue waspurified by flash column chromatography, eluting with ethylacetate/chloroform (1:19), to provide Intermediate 13 as a light yellowsolid (0.88 g, 20%): TLC R_(f) (1:9 ethyl acetate/chloroform)=0.60; ¹HNMR (300 MHz, DMSO-d₆): δ 10.48 (s, 1H), 7.42 (s, 1H), 7.37 (d, J=10.0Hz, 1H), 7.09 (d, J=9.9 Hz, 1H), 6.90 (d, J=6.2 Hz, 2H), 6.83 (s, 1H),6.00 (s, 2H), 5.15 (s, 1H), 4.16 (q, J=6.3 Hz, 2H), 3.85-3.80 (m, 1H),2.90-2.80 (m, 1H), 1.24 (t, J=6.9 Hz, 3H). The trans-β-carboline alsowas obtained as a light yellow solid, but was not characterized (0.60 g,14%): TLC R_(f) (1:9 ethyl acetate/chloroform)=0.33.

Intermediate 14

Preparation of a cis-2-chloroacetyl-β-carboline

Chloroacetyl chloride (0.2 mL, 2.6 mmol) was added dropwise to asolution of Intermediate 13 (0.88 g, 2.0 mmol) and triethylamine (0.4mL, 2.6 mmol) in methylene chloride (10 mL) at 0° C. under a nitrogenblanket. The mixture was slowly warmed to room temperature and stirredfor 5 hours. The resulting white suspension was diluted with methylenechloride (100 mL), washed with brine (100 mL), dried over Na₂SO₄, andfiltered. The solvent was removed under reduced pressure to provideIntermediate 14 as an amber foam, which was used without furtherpurification (1.03 g): TLC R_(f) (1:9 ethyl acetate/chloroform)=0.76.

Intermediate 16

A mixture of crude Intermediate 14 (1.0 g, 1.9 mmol) and methylamine (4mL, 7.7 mmol, 2.0 M in THF) in methanol (10 mL) was heated at refluxunder a nitrogen blanket for 2 hours, after which the resulting orangesuspension was cooled to room temperature. The solids were collected byvacuum filtration and dried in a vacuum oven at 60° C. overnight toprovide Intermediate 15 as a white powder (0.45 g, 49% over two steps):mp 324-330° C.; TLC R_(f) (1:9 methanol/chloroform)=0.78; ¹H NMR (300MHz, DMSO-d₆): δ 11.18 (s, 1H), 7.53 (d, J=8.4 Hz, 1H), 7.49 (s, 1H),7.14 (d, J=8.3 Hz, 1H), 6.86 (s, 1H), 6.78 (s, 2H), 6.11 (s, 1H), 5.93(s, 2H), 4.39 (dd, J=4.4, 11.5 Hz, 1H), 4.17 (d, J=17.1 Hz, 1H), 3.95(d, J=17.2 Hz, 1H), 3.52 (dd, J=4.5, 15.9 Hz, 1H), 3.01 (d, J=11.7 Hz,1H), 2.93 (s, 3H); API MS m/z 470 [C₂₂H₁₈BrN₃O₄+H]⁺. Anal. Calcd. forC₂₂H₁₈BrN₃O₄: C, 56.42; H, 3.87; N, 8.97. Found: C, 56.34; H, 3.92; N,8.82. The sterochemistry of Intermediate 15 was confirmed to be thedesired cis isomer by a series of NOE difference experiments: positiveNOE enhancements from the C12a proton at 4.39 ppm to the C6 proton at6.11 ppm and a C12 proton at 3.52 ppm; a positive NOE enhancement fromthe C6 proton at 6.11 ppm to the C12a proton at 4.39 ppm.

EXAMPLE 4

Preparation of (+−,cis)-6-Benzo[1,3]dioxol-5-yl-2-methyl-9-phenyl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione

Phenyl boronic acid (0.09 g, 0.74 mmol) was added to a degassed mixtureof Intermediate 16 (0.172 g, 0.37 mmol), barium hydroxide octahydrate(0.58 g, 1.85 mmol), and bis(triphenylphosphine)palladium (II) chloride(0.05 g, 0.07 mmol) in ethanol (10 mL), and the resulting mixture wasstirred at room temperature for 48 hours. An additional portion ofphenyl boronic acid (0.09 g, 0.74 mmol) then was added, after which themixture was stirred at room temperature for an additional 12 hours. Thepalladium catalyst was removed by vacuum filtration through a plug ofCelite, and the resulting filtrate was concentrated under reducedpressure and purified by flash column chromatography, eluting withmethylene chloride/ethyl acetate (4:1), to provide a white solid. Thissolid was further purified by recrystallization from methylene chloridefollowed by vacuum filtration to yield a solid, which was driedovernight under vacuum at 60° C. to provide Example 4 as a white solid(0.049 g, 29%): mp 198-208° C.; TLC R_(f) (1:4 ethylacetate/chloroform)=0.37; ¹H NMR (500 MHz, CDCl₃): δ 7.91 (s, 1H), 7.46(s, 1H), 7.37 (d, J=8.4 Hz, 2H), 7.27-7.25 (m, 4H), 6.95 (s, 1H), 6.79(s, 1H), 6.72-6.67 (m, 2H), 5.93 (s, 2H), 5.29 (s, 1H), 4.34 (dd, J=4.0,4.11 Hz, 1H), 4.12 (dd, J=1.2, 1.1 Hz, 1H), 3.51 (dd, J=4.2, 4.2 Hz,1H), 3.00 (s, 3H), 2.93-2.88 (m, 2H) ppm; API MS m/z 467[C₂₈H₂₃N₃O₄+H]⁺. HPLC analysis (Symmetry C18 Column, 150×3.5 mm.Retention Time=10.9 minutes; 30/0.85:70/0.1 acetonitrile/TFA:water/TFAto 100/0.85 acetonitrile/TFA over 20 minutes; flow=1.0 mL/min; detectorat 220 nm; 25° C.) showed one peak, with a purity of 91.5%. Chiral HPLCanalysis (Chiralcel OD Column, 250×4.6 mm, Retention Times=12.9 and 17.4min; 1:1 isopropanol/hexanes; flow=0.5 mL/min; detector at 222 nm; 25°C.) showed two major peaks, with a ratio of 54:42 and with a totalpurity of 96.6%. The relative stereochemistry of Intermediate 4 wasconfirmed to be the desired cis isomer by a series of NOE differenceexperiments: a positive NOE enhancement from the C12a proton at 4.34 ppmto the C6 proton at 6.79 ppm; a positive NOE enhancement from the C6proton at 6.79 ppm to the C12a proton at 4.34 ppm.

PREPARATION OF EXAMPLE 5

Example 5 was prepared from indole-6-carboxylic acid as depicted in thefollowing synthetic scheme. Indole-6-carboxylic acid is a commerciallyavailable compound. Aspects of the following synthetic scheme aredisclosed in Y. Yokoyama et al., Tetrahydron Lett, 40, p. 7803 (1990);and H.R. Snyder et al., J. Am Chem. Soc., 77, p. 1257 (1955).

Intermediate 17Preparation of N-Acetyl-6-carboxytryptophan

A mixture of indole-6-carboxylic acid (5.0 g, 31 mmol), DL-serine (3.25g, 31 mmol), and acetic anhydride (8.8 mL, 93 mmol) in glacial aceticacid (50 mL) was heated under a nitrogen blanket at 80° C. for 24 hours.The resulting brown solution was cooled to room temperature, then thesolvent was removed under reduced pressure to provide Intermediate 17 asa brown foam, which was used without further purification (11.0 g): ¹HNMR (300 MHz, D₂O): δ 8.08 (s, 1H), 7.72 (s, 2H), 7.40 (s, 1H),4.61-4.53 (m, 1H), 3.45-3.35 (m, 1H), 3.23-3.13 (m, 1H), 1.92 (s, 3H)ppm.

Intermediate 18

Preparation of 6-Carboxytryptophan

A solution of crude Intermediate 17 (11.0 g) was heated in 15% aqueoussodium hydroxide for 18 hours. The reaction mixture was cooled to 0° C.,then acidified to pH 5 with acetic acid, after which the solution wasconcentrated under reduced pressure. The residue was slurried in water(500 mL) then the solids were collected by filtration under reducedpressure and dried in a vacuum oven at 100° C. for 18 hours to yieldIntermediate 18 as a gray solid (2.6 g, 34% over two steps). A secondportion of Intermediate 18 was recovered from the filtrate (0.75 g, 10%over two steps): ¹H NMR (300 MHz, DMSO-d₆): δ 11.30 (s, 1H), 8.00 (s,1H), 7.68-7.52 (m, 2H), 7.40 (s, 1H), 3.50-3.40 (m, 1H), 3.39-3.25 (m,1H), 3.10-2.92 (m, 1H) ppm.

Intermediates 19-21

Preparation of 6-Methylcarboxytryptophan

Thionyl chloride (0.84 mL, 11 mmol) was added dropwise to a suspensionof Intermediate 18 (0.75 g, 302 mmol) in methanol (15 mL) at 0° C. undera nitrogen blanket, then the mixture was warmed to room temperature andstirred for 20 hours. The resulting solution was diluted with ethylacetate (200 mL), extracted with saturated sodium bicarbonate (NaHCO₃)solution (2×50 mL) and acidified to pH 6 with acetic acid. The resultingslurry was filtered, then the solid was washed with several smallportions of water, and dried in a vacuum oven at room temperature toyield a mixture of Intermediates 19 and 20 as a brown solid (400 mg,51%).

Thionyl chloride (0.45 mL, 6.0 mmol) was added dropwise to a slurry ofIntermediates 19 and 20 (316 mg, 1.21 mmol) in methanol (15 mL) at 0° C.under a nitrogen blanket. This mixture was warmed to room temperatureand stirred for 3 hours, then the mixture was heated to 50° C. to stirfor an additional 2 hours. The solvent was removed under reducedpressure, and the residue was diluted with ethyl acetate (200 mL). Thesolution was washed with saturated NaHCO₃ (20 mL), water (20 mL), andbrine (20 mL), dried over Na₂SO₄, filtered, and the solvent was removedunder reduced pressure to yield Intermediate 21 as a brown oil (310 mg,93%): TLC R_(f) (5:1 methylene chloride/ethyl acetate)=0.1: ¹H NMR (300MHz, CDCl₃): δ 9.11 (bs, 1H), 8.07 (s, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.61(d, J=8.0 Hz, 1H), 7.19 (s, 1H), 3.93 (s, 3H), 3.90-3.82 (m, 1H), 3.71(s, 1H), 3.31-3.22 (m, 1H), 3.13-3.05 (m, 1H) ppm.

Intermediate 22

Preparation of 5-Carboline

A mixture of Intermediate 21 (495 mg, 1.79 mmol), piperonal (479 mg, 3.2mmol), and p-toluene sulfonic acid monohydrate (80 mg) was refluxed intoluene with water removal via Dean-Stark trap for 5 hours under anitrogen blanket. The dark brown mixture was cooled to room temperatureand diluted with ethyl acetate (250 mL). The mixture then was washedsuccessively with saturated NaHCO₃ (30 mL), water (20 mL), and brine (20mL), dried over Na₂SO₄, filtered, and the solvent was removed underreduced pressure. The residue was slurried in a mixture of methylenechloride/ethyl acetate (5:1, 10 mL) and the slurry was filtered underpressure to yield a mixture of the cis- and trans- Intermediate 22,which were used without characterization (311 mg, 42%).

Intermediate 23

Preparation of cis-2-Chloroacetyl-β-carboline

Chloroacetyl chloride (0.076 mL, 0.99 mmol) was added dropwise to amixture of Intermediate 22 (311 mg, 0.76 mmol, mixture of cis and transisomers) and triethylamine (0.138 mL, 0.99 mmol) in chloroform (10 mL)at 0° C. under a nitrogen blanket. The resulting mixture was stirred at0° C. for 1 hour, then warmed to room temperature and stirred for anadditional 2 hours. The solution was diluted with ethyl acetate (100mL), washed with saturated NaHCO₃ solution (10 mL), water (10 mL), andbrine (10 mL), dried over sodium sulfate (Na₂SO₄), and the solvent wasremoved under reduced pressure to provide a yellow foam. The mixture waspurified by flash column chromatography, eluting with hexanes/ethylacetate (2:1), to yield Intermediate 23 as a white solid, which was notcharacterized (195 mg, 53%): TLC R_(f) (2:1 hexanes/ethyl acetate)=0.62.The latter eluting trans-2-chloroacetyl-β-carboline was obtained as awhite solid which also was not characterized (160 mg, 43%): TLC R_(f)(2:1 hexanes/ethyl acetate)=0.40.

EXAMPLE 5

Preparation of (+−,cis)-6-Benzo[1,3]dioxol-5-yl-2-methyl-1,4-dioxo-1,2,3,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-9-carboxylicacid methyl ester

A mixture of Intermediate 23 (132 mg, 0.27 mmol) and methylamine (6.2mL, 12.4 mmol, 2 M solution in THF) in methylene chloride (18 mL) wasrefluxed under a nitrogen blanket for 4 hours. The resulting solutionwas concentrated under reduced pressure to yield a yellow solid, whichwas stirred in methanol (4 mL) for 1 hour. The solid was isolated byfiltration under reduced pressure, washed with methanol (5×1 mL), anddried in a vacuum oven at 70° C. for 17 hours to provide Example 5 as anoff-white solid (117 mg, 96%): mp 294-295° C.; TLC R_(f) (5:1:0.5methylene chloride/ethyl acetate/methanol)=0.65; ¹H NMR (300 MHz,CDCl₃): δ 8.25 (bs, 1H), 8.03 (s, 1H), 7.84 (d, J=8.7 Hz, 1H), 7.60 (d,J=8.7 Hz, 1H), 6.86 (dd, J=8.0, 1.7 Hz, 1H), 6.73 (d, J=1.7 Hz, 1H),6.69 (d, J=8.0 Hz, 1H), 6.20 (s, 1H), 5.88 (s, 1H), 5.86 (s, 1H), 4.30(dd, J=11.6, 4.5 Hz, 1H), 4.11 (dd, J=17.6, 1.3 Hz, 1H), 4.00-3.88 (m,4H), 3.78 (dd, J=16.1, 4.5 Hz, 1H), 3.27-3.16 (m, 1H), 3.05 (s, 3H) ppm;CI MS m/z 448 [C₂₄H₂₁N₃O₆+H]⁺, Anal. Calcd. for C₂₄H₂₁N₃O₆: C, 64.42; H,4.73; N, 9.39. Found: C, 64.12; H, 4.61; N, 9.12. HPLC analysis (AquasilC18 Column, 100×4.6 mm, Retention Time=10.3 minutes; 45:55/0.03acetonitrile:water/TFA; flow=0.50 mL/min.; detector @ 254 nm;temperature ambient) showed one peak, with a purity of 99.7%. Thestereochemistry of Example 5 was confirmed to be the cis isomer by aseries of NOE difference experiments: a positive NOE enhancement fromthe C12a proton at 4.30 ppm to the C6 proton at 6.20 ppm; a positive NOEenhancement from the C6 proton at 6.20 ppm to the C12a proton at 4.30ppm.

PREPARATION OF EXAMPLE 6

(+−,trans)-6-Benzo[1,3]dioxol-5-yl-2-methyl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-9-carobxylicacid

Example 6 was prepared from Example 5 by the following procedure.

A mixture of Example 5 (0.125 g, 0.28 mmol), 2 N sodium hydroxidesolution (2 mL), THF (6 mL), and methanol (2 mL) was heated at 80° C.for 24 hours. The resulting yellow solution was cooled to roomtemperature and diluted with water, then the solution was acidified topH 6 with acetic acid. The resulting solution was concentrated underreduced pressure to a volume of 20 mL, and was allowed to standovernight at room temperature. The resulting solid precipitate wasisolated by filtration under reduced pressure, then further purified bystirring in diethyl ether (4 mL) at 40° C. for 2 hours. The resultingsolids were isolated by filtration under reduced pressure, washed withdiethyl ether (5×1 mL), and dried in a vacuum oven at 90° C. for 4 hoursto provide Example 6 as an off-white solid (0.093, 78%): mp 246-252° C.;¹H NMR (300 MHz, CD₃OD): δ 8.05 (s, 1H), 7.76 (d, J=8.6 Hz, 1H), 7.56(d, J=8.14 Hz, 1H), 6.99 (s, 1H), 6.83-6.73 (m, 2H), 6.66 (dd, J=8.0 Hz,1H), 5.93 (s, 2H), 4.34-4.23 (m, 2H), 4.08 (d, J=17.3 Hz, 1H), 3.44 (dd,J=15.4, 4.2 Hz, 1H), 3.08-2.95 (m, 4H) ppm; ESI MS m/z 432[C₂₃H₁₉N₃O₆—H]⁺. Anal. Calcd for C₂₃H₁₉N₃O₆: C, 63.74; H, 4.42; N, 9.70.Found: C, 62.77; H, 4.61; N, 9.19. HPLC analysis (Aquasil C18 Column,100×4.6 mm, Retention Time=25.4 minutes; 30:70/0.03acetonitrile:water/TFA; flow=0.50 mL/min; detector at 254 nm;temperature ambient) showed one peak, with a purity of 98.9%. Thestereochemistry of Example 6 was confirmed to be the trans isomer by aseries of NOE difference experiments: no NOE enhancement from the C12aproton at 4.30 ppm to the C6 proton at 6.99 ppm; no NOE enhancement fromthe C6 proton at 6.99 ppm to the C12a proton at 4.30 ppm.

PREPARATION OF EXAMPLE 7

Example 7 was prepared from 6-cyanoindole as depicted in the followingsynthetic scheme. 6-Cyanoindole is a commercially available compound

Intermediate 24Preparation of Gramine

Acetic acid (6 ml) was added dropwise to aqueous dimethylamine (7.0 ml,55.7 mmol) at a rate that maintained the solution below 5° C. To thismixture was added aqueous formaldehyde (4.2 ml, 55.7 mmol) dropwise. Theresulting mixture then was added to a solution of 6-cyanoindole (6.6 g,46.4 mmol) in acetic acid (30 ml) over 15 minutes at room temperatureunder a nitrogen blanket. The resulting dark yellow solution was stirredat room temperature for 3 hours, followed by dilution with 2 N sodiumhydroxide (30 ml), then stored at 0° C. for 12 hours. The solution wasdecanted, and the solvent was removed under reduced pressure to producea white foam. The white foam was dissolved in water (200 mL), andadjusted to a basic pH with a saturated NaHCO₃ solution. The resultingmixture was extracted with ethyl acetate (3×200 mL), dried over Na₂SO₄,and the solvent was removed under reduced pressure to yield Intermediate24 as an off-white solid (6.24 g, 65%): ¹H NMR (300 MHz, DMSO-d₆): δ11.52 (s, 1H), 7.85 (s, 1H), 7.77 (d, J=8.2 Hz, 1H), 7.54 (d, J=2.4 Hz,1H), 7.31 (dd, J=1.5, 8.3 Hz, 1H), 3.56 (s, 2H), 2.13 (s, 6H) ppm.

Intermediate 25

Preparation of Diester

To a solution of diethyl formamidomalonate (6.61 g, 32.5 mmol) inethanol (40 mL) under an argon blanket was added sodium ethoxide (12.0mL, 32.5 mmol, 2.7 M solution in ethanol). The resulting mixture wasstirred at room temperature for 30 minutes to yield a slurry. To theslurry was added Intermediate 24 (5.39 g, 27.1 mmol), followed bydimethyl sulfate (5.1 mL, 54.2 mmol). The resulting clear brown solutionwas stirred at room temperature, and after 15 minutes a precipitateformed. The resulting slurry was stirred at room temperature for 16hours. Then the solids were removed by filtration under reducedpressure, and the filtrate concentrated to yield diethyl esterIntermediate 25 as an orange solid which was used without furtherpurification (10.9 g): TLC R_(f) (4:1 methylene chloride/ethylacetate)=0.50.

Intermediate 26

Preparation of Amino Diester

To a solution of Intermediate 25 (27.1 mmol) in methanol (120 mL) wasadded hydrochloric acid in diethyl ether (54 mL, 54.2 mmol, 1 M solutionin diethyl ether). The resulting solution was allowed to sit overnightunder an argon blanket. The mixture then was concentrated under reducedpressure, diluted with ethyl acetate (100 mL), and extracted with 2 NHCl (3×200 mL). The combined aqueous extracts were adjusted to pH 8 withNa₂CO₃, followed by extraction with methylene chloride (3×200 mL). Thecombined organic extracts were dried over Na₂SO₄, and the solvent wasremoved under reduced pressure to provide Intermediate 26 as an orangeoil (2.57 g, 30%): ¹H NMR (300 MHz, CDCl₃): δ 8.79 (bs, 1H), 7.69-7.64(m, 2H), 7.34-7.30 (m, 2H), 4.27-4.15 (m, 4H), 3.48 (s, 2H), 1.99 (bs,2H), 1.31-1.23 (m, 6H) ppm.

Intermediate 27

Preparation of (+/−)-6-Cyanotryptophan Ethyl Ester

To a mixture of Intermediate 26 (2.57 g, 7.80 mmol) in ethanol (80 mL)was added a solution of sodium hydroxide (0.37 g, 9.25 mmol) in water (5mL). The resulting orange solution was stirred at room temperature for16 hours, after which an additional 0.5 equivalents of sodium hydroxidein water was added. The mixture was stirred for 5 hours, then acidifiedto pH 6 with acetic acid. Concentration of the solution under reducedpressure provided an oil which was dissolved in 2:1 methylenechloride/methanol (60/20 mL), then heated at reflux for 5 hours. Theresulting slurry was cooled, then the solids were removed by filtrationunder reduced pressure. The filtrate was concentrated, dissolved inwater (100 mL), then extracted with methylene chloride (3×100 mL). Theorganic extracts were dried over Na₂SO₄, and the solvent was removedunder reduced pressure to provide Intermediate 27 as an orange oil (0.91g, 46%): ¹H NMR (500 MHz, DMSO-d₆): δ 11.40 (s, 1H), 7.81 (s, 1H), 7.68(d, J=10.0 Hz, 1H), 7.45 (s, 1H), 7.30 (d, J=10.0 Hz, 1H), 4.00-3.96 (m,2H), 3.60-3.57 (m, 1H), 3.03-2.94 (m, 2H), 2.08 (bs, 2H), 1.10-1.07 (m,3H) ppm.

Intermediate 28

Preparation of (+/−)-cis-β-Carboline

A solution of Intermediate 27 (0.49 g, 1.9 mmol), piperonal (0.37 g, 2.5mmol) and p-toluene-sulfonic acid monohydrate (0.050 g, 0.26 mmol) inxylenes (30 mL) was heated at 145° C. under an argon blanket for 16hours with water removal via Dean-Stark trap. The resulting orangesolution was cooled, diluted with methylene chloride (10.0 mL), thenwashed with saturated NaHCO₃ (20 mL). The organic layer was dried overNa₂SO₄, then the solvent was removed under reduced pressure to providean orange oil. The residue was purified by flash column chromatography,eluting with methylene chloride/ethyl acetate (6:1) to provideIntermediate 28 as orange solid (0.20 g, 27%),: TLC R_(f) (5:1 methylenechloride/ethyl acetate)=0.59; ¹H NMR (300 MHz, DMSO-d₆): δ 10.96 (s, H),7.63-7.59 (m, 2H), 7.30 (d, J=9.9 Hz, 1H), 6.94-6.84 (m, 0.3H), 6.01 (s,2H), 5.20 (d, J=9.9 Hz, 1H), 4.19 (q, J=7.3 Hz, 2H), 3.87-3.82 (m, 1H),3.12-3.03 (m, 1H), 2.89-2.78 (m, 1H), 2.28 (t, J=7.1 Hz, 3H) ppm. Thelatter eluting trans isomer also was isolated as an orange solid (0.17g, 23%): TLC R_(f) (5:1 methylene chloride/ethyl acetate)=0.28; ¹H NMR(300 MHz, DMSO-d₆): δ 11.21 (s, 1H), 7.69 (s, 1H), 7.63 (d, J=9.9 Hz,1H), 7.30 (d, J=9.9 Hz, 1H), 6.88-6.82 (m, 2H), 6.68 (d, J=9.0 Hz, 1H),5.98 (s, 2H), 5.31 (s, 1H), 4.12-4.05 (m, 2H), 3.84-3.79 (m, 1H),3.14-3.03 (dd, J=5.0, 15.0 Hz, 1H), 2.95-2.88 (dd, J=8.0, 150 Hz, 1H),1.18 (t, J=7.5 Hz, 3H) ppm.

Intermediate 29

Preparation of (+/−)-cis-2-Chloroacetyl-β-Carboline

Chloroacetyl chloride (0.055 mL, 69 mmol) was added to a solution ofIntermediate 28 (0.20 g, 0.51 mmol) and triethylamine (0.10 mL, 0.72mmol) in methylene chloride (10 mL) at 0° C. under an argon blanket,after which the solution was warmed to room temperature over 2 hours.The yellow solution then was diluted with methylene chloride (40 mL),washed with water (20 mL), and saturated NaHCO₃ solution (20 mL). Theorganic layer was dried over Na₂SO₄, and the solvent was removed underreduced pressure to afford Intermediate 29 as a yellow oil which wasused without further purification: TLC R_(f) (8:1 methylenechloride/ethyl acetate)=0.76.

EXAMPLE 7

Preparation of (+−,cis)-6-Benzo[1,3]dioxol-5-yl-2-methyl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-9-carbonitrile

A solution of cis-2-chloroacetyl-β-carboline 7 (0.51 mmol) andmethylamine (5.1 mL, 10.2 mmol, 2 M solution in THF) in methanol (9.0mL) was heated at 50° C. for 16 hours. The resulting solids wereisolated by filtration under reduced pressure to provide Example 7 as apale yellow solid (94 mg. 44%): mp 329-331° C.; TLC R_(f) (5:1:0.5methylene chloride/ethyl acetate/methanol)=0.50; ¹H NMR (300 MHz,DMSO-d₆): δ 11.65 (s, H), 7.81 (s, 1H), 7.76 (d, J=8.2 Hz, 1H), 7.33 (d,J=8.4 Hz, 1H), 6.88 (s, 1H), 6.79 (s, 2H), 6.17 (s, 2H), 5.93 (s, 2H),4.43-4.38 (dd, J=4.1, 11.5 Hz, 1H), 4.17 (d, J=17.3 Hz, 1H), 3.95 (d,J=17.3 Hz, 1H), 3.59 (dd, J=4.4, 16.0 Hz, 1H), 3.00 (dd, J=11.5, 15.7Hz, 1H), 2.93 (s, 3H) ppm; ESI MS m/z 413 [C₂₃H₁₈N₄O₄—H]⁺. Anal. Calcd.for C₂₃H₁₈N₄O₄; C, 66.66; H, 4.38; N, 13.52. Found: C, 66.49; H, 4.43;N, 13.30. HPLC analysis (Aquasil C18 Column, 100×4.6 mm, RetentionTime=9.74 min; 45:55 0.03% acetonitrile:water/TFA; flow=0.50 mL/min;detector @ 254 nm; temperature ambient) showed one peak, with a purityof 100.0%. The stereochemistry of analog Example 7 was confirmed to bethe desired cis isomer by a series of NOE difference experiments: apositive NOE enhancement from the C12a proton at 4.40 ppm to the C6proton at 6.17 ppm; a positive NOE enhancement from the C6 proton at6.17 ppm to the C12a proton at 4.40 ppm.

PREPARATION OF EXAMPLE 8

Example 8 was prepared from 3-methyl-2-nitrophenol as depicted in thefollowing synthetic scheme. Intermediate 31 was prepared as disclosed inG. M. Carrera et al., Synlet, p. 93 (1994) and M. P. Moyer et al., J.Org. Chem., 51, p. 5106 (1986), and converted to Intermediate 32 asdisclosed in Y. Yokohama, Tetrahedron Lett., 40, p. 7803 (1999) and H.R. Snyder et al., J. Am. Chem. Soc., 77, p. 1257 (1955).

Intermediate 31Preparation of 7-Benzyloxyindole

Benzyl bromide (15.4 ml, 130 mmol) was added dropwise to a solution of3-methyl-2-nitrophenol (20.0 g, 130 mmol) and sodium hydroxide (6.0 g,144 mmol) in anhydrous N,N-dimethylformamide (100 mL) at 0° C. under anitrogen blanket. The resulting red solution was slowly warmed to roomtemperature, stirring for a total of 17 hours. The mixture then wasdiluted with ethyl acetate (706 ml), stirred for 15 minutes, and thesolids were removed by filtration under reduced pressure. The brightyellow filtrate was concentrated under reduced pressure to provideIntermediate 30 as a dark yellow residue, which was used without furtherpurification (31.8 g): TLC R_(f) (5:1 hexanes/ethyl acetate)=0.50.

A mixture of Intermediate 30 (31.8 g, 130 mmol), pyrrolidine (11.0 mL,130 mmol), and N,N-dimethylformamide dimethyl acetal (52 mL, 392 mmol)in anhydrous N,N-dimethylformamide (100 mL) was heated at 110° C. undera nitrogen blanket for 3 hours. The cooled mixture was diluted withwater (1.5 L), extracted with ether (2×500 mL), dried over Na₂SO₄, andfiltered. The solvent was removed under reduced pressure to afford theintermediate enamine as a dark red oil, which was immediately used inthe next step without purification.

A solution of the enamine in methanol (150 mL) and acetic acid (5 mL)was treated with a catalytic amount of 10% palladium on carbon (2 g, 10%wet), and the resulting mixture was stirred under a hydrogen atmosphere(50 psi) at room temperature for 2 hours. The palladium catalyst wasremoved by vacuum filtration through MgSO₄ (10 g), and the filtrate wasconcentrated under reduced pressure to provide a dark brown residue. Theresidue was purified by flash column chromatography, eluting with ethylacetate/chloroform (1:19), to provide Intermediate 31 as an off-whitesolid (3.82 g, 21% over three steps): TLC R_(f) (4:1 hexanes/ethylacetate)=0.61; ¹H NMR (300 MHz, DMSO-d₆): δ 11.18 (s, 1H), 7.56 (d,J=7.2 Hz, 2H), 7.43-7.38 (m, 3H), 7.35 (s, 1H), 7.23 (d, J=2.9 Hz, 1H),6.88 (t, J=7.6 Hz, 1H), 6.72 (d, J=7.6 Hz, 1H), 6.39 (t, J=2.5 Hz, 1H),5.26 (s, 2H) ppm.

Intermediate 32

Preparation of (+/−)-7-Benzyloxytryptophan

Acetic anhydride (0.5 mL, 5.2 mmol) was added to a slurry ofIntermediate 31 (0.575 g, 2.6 mmol) and DL-serine (0.270 g, 2.6 mmol) inanhydrous acetic acid (5 mL) at room temperature under a nitrogenblanket. The resulting orange solution was heated to reflux and stirredfor 24 hours. The cooled mixture then was concentrated under reducedpressure to provide the intermediate N-acetyltryptophan as dark red oil,which was used immediately without purification.

A suspension of the intermediate N-acetyltryptophan in 3 N sodiumhydroxide was heated at reflux for 17 hours, then cooled to roomtemperature. The mixture was diluted with water (50 mL), washed withdiethyl ether (50 mL), slurried with charcoal (1 g), and filteredthrough a plug of silica gel (20 g), eluting with water (100 mL). Thefiltrate was acidified to pH 4 with 6 N HCl (1 mL), and the solution wasfiltered through a plug of DOWEX 50×8-100 ion-exchange resin (100 g),eluting with concentrated ammonium hydroxide/methanol (1:3), to provideIntermediate 32 as a gray powder (0.040 g, 5% over two steps): ¹H NMR(300 MHz, CD₃OD): δ 7.51 (d, J=7.0 Hz, 2H), 7.39-7.29 (m, 3H), 7.17 (d,J=7.9 Hz, 1H), 7.04 (s, 1H), 6.91 (t, J=7.6 Hz, 1H), 6.68 (d, J=7.7 Hz,1H), 5.21 (s, 2H), 4.72-4.68 (m, H), 3.34-3.29 (m, 1H), 3.16-3.09 (m,1H) ppm.

Intermediate 33

Preparation of (+/−)-7-Benzyloxytryptophan Methyl Ester Hydrochloride

Thionyl chloride (0.4 mL, 4.8 mmol) was added to a suspension ofIntermediate 32 (0.600 g, 1.9 mmol) in anhydrous methanol (10 mL) at 0°C. under a nitrogen blanket. The resulting mixture was heated to 50° C.and stirred for 1 hour, then cooled to room temperature. The resultingdark green solution was concentrated under reduced pressure to provideIntermediate 33 as a light green powder which was used without furtherpurification (0.710 g, 100%): TLC R_(f) (4:1 chloroform/methanol)=0.87;¹H NMR (300 MHz, DMSO-d₆): δ 11.18 (s, 1H), 8.48 (bs, 2H), 7.55 (d,J=7.2 Hz, 1H), 7.43-7.41 (m, 3H), 7.38 (s, 1H), 7.34 (d, J=7.2 Hz, 1H),7.10-6.91 (m, 1H), 6.75 (d, J=7.6 Hz, 1H), 5.26 (s, 2H), 4.42 (s, 1H),3.66 (s, 3H), 3.35-3.26 (m, 2H) ppm.

Intermediate 34

Preparation of (+/−)-cis-β-Carboline

A solution of Intermediate 33 (0.710 g, 2.0 mmol) and piperonal (0.300g, 2.0 mmol) in anhydrous isopropanol (10 mL) was heated at reflux undera nitrogen blanket for 6 hours. The resulting orange solution was cooledto room temperature, neutralized with saturated NaHCO₃ solution (1 mL),then the solvent was removed under reduced pressure to provide brownsolid. The residue was purified by flash column chromatography, elutingwith chloroform/ethyl acetate (19:1), to provide Intermediate 34 as acolorless oil (0.120 g, 13%): TLC R_(f) (4:1 chloroform/ethylacetate)=0.57; ¹H NMR (300 MHz, CDCl₃): δ 7.70 (s, 1H), 7.44-7.26 (m,5H), 7.17 (d, J=7.8 Hz, 1H), 7.04 (t, J=7.7 Hz, 1H), 6.88-6.70 (m, 4H),5.94 (s, 2H), 5.16 (s, 2H), 3.93 (dd, J=4.1, 11.0 Hz, 1H), 3.81 (s, 3H),3.22-3.17 (m, 1H), 3.03-2.95 (m, 1H) ppm. The latter eluting transisomer was also isolated as a colorless oil, but was not characterized(0.190 g, 21%) TLC R_(f) (4:1 chloroform/ethyl acetate)=0.51.

Intermediate 35

Preparation of (+/−)-cis-2-Chloroacetyl-β-carboline

Chloroacetyl chloride (0.03 mL, 0.3 mmol) was added to a solution ofIntermediate 34 (0.120 g, 0.3 mmol) and triethylamine (0.05 mL, 0.3mmol) in methylene chloride (5mL) at 0° C. under an argon blanket, thenwarmed to room temperature over 1 hour. The solvent then was removedunder reduced pressure to yield Intermediate 35 as a yellow powder whichwas used without further purification: TLC R_(f) (4:1 chloroform/ethylacetate)=0.71.

EXAMPLE 8

Preparation of (+−,cis)-6-Benzo[1,3]dioxol-5-yl-8-benzyloxy-2-methyl-2,3,6,7,12,12a-hexahydropyrazino[1′2′:1,6]pyrido[3,4-b]indole-1,4-dione

A solution of Intermediate 35 (0.3 mmol) and methylamine (0.7 mL, 1.2mmol, 2 M solution in THF) in methanol (5 mL) was heated at 50° C. for20 hours. The resulting solids were isolated by filtration under reducedpressure to provide Example 8 as a pale yellow powder (0.092 g, 71% overtwo steps): mp 150-161° C.; TLC R_(f) (4:1 chloroform/ethylacetate)=0.19; ¹H NMR (300 MHz, CDCl₃): δ 8.25 (s, H), 7.45-7.34 (m,6H), 7.26-7.22 (m, 1H), 7.06 (t, J=7.9 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H),6.74-6.65 (m, 2H), 6.17 (s, 1H), 5.86 (s, 2H), 5.14 (s, 2H), 4.23 (dd,J=4.3, 11.5 Hz, 1H), 3.95 (dd, J=17.4, 52.8 Hz, 2H), 3.75 (dd, J=4.5,11.4 Hz, 1H), 3.19 (dd, J=4.3, 11.7 Hz, 1H), 3.01 (s, 3H) ppm; ESI MSm/z 496 [C₂₉H₂₅N₃O₅+H]⁺. Anal. Calcd. for C₂₉H₂₅N₃O₅: C, 70.29; H, 5.09;N, 8.48. Found: C, 69.71; H, 5.09; N, 8.41. The stereochemistry ofExample 8 was confirmed to be the desired cis isomer by a series of NOEdifference experiments: a positive NOE enhancement from the C12a protonat 4.23 ppm to the c6 proton at 6.17 ppm; a positive NOE enhancementfrom the C6 proton at 6.17 ppm to the C12a proton at 4.23 ppm.

The following compounds are additional examples of compounds ofstructural formula (I) that can be prepared by methods analogous to thepreparation of Examples 1 through 8.

EXAMPLE 9

EXAMPLE 10

EXAMPLE 11

EXAMPLE 12

EXAMPLE 13

EXAMPLE 14

Compounds of the present invention can be formulated into tablets fororal administration. For example, a compound of formula (I) can beformed into a dispersion with a polymeric carrier by the coprecipitationmethod set forth in WO 96/38131, incorporated herein by reference. Thecoprecipitated dispersion can be blended with excipients, then pressedinto tablets, which optionally are film-coated.

The compounds of structural formula (I) were tested for an ability toinhibit PDE5. The ability of a compound to inhibit PDE5 activity isrelated to the IC₅₀ value for the compound, i.e., the concentration ofinhibitor required for 50% inhibition of enzyme activity. The IC₅₀ valuefor compounds of structural formula (I) were determined usingrecombinant human PDE5.

The compounds of the present invention typically exhibit an IC₅₀ valueagainst recombinant human PDE5 of less than about 50 μM, and preferablyless than about 25 μM, and more preferably less than about 15 μm. Thecompounds of the present invention typically exhibit an IC₅₀ valueagainst recombinant human PDE5 of less than about 1 μM, and often lessthan about 0.05 μM. To achieve the full advantage of the presentinvention, a present PDE5 inhibitor has an IC₅₀ of about 0.1 nM to about15 μM.

The production of recombinant human PDEs and the IC₅₀ determinations canbe accomplished by well-known methods in the art. Exemplary methods aredescribed as follows:

Expression of Human PDEs

Expression in Saccharomyces cerevisiae (Yeast)

Recombinant production of human PDE1B, PDE2, PDE4A, PDE4B, PDE4C, PDE4D,PDE5, and PDE7 was carried out similarly to that described in Example 7of U.S. Pat. No. 5,702,936, incorporated herein by reference, exceptthat the yeast transformation vector employed, which is derived from thebasic ADH2 plasmid described in Price et al., Methods in Enzymology,185, pp. 308-318 (1990), incorporated yeast ADH2 promoter and terminatorsequences and the Saccharomyces cerevisiae host was theprotease-deficient strain BJ2-54 deposited on Aug. 31, 1998 with theAmerican Type Culture Collection, Manassas, Va., under accession numberATCC 74465. Transformed host cells were grown in 2×SC-leu medium, pH6.2, with trace metals, and vitamins. After 24 hours, YEPmedium-containing glycerol was added to a final concentration of2×YET/3% glycerol. Approximately 24 hr later, cells were harvested,washed, and stored at −70° C.

Human Phosphodiesterase Preparations

Phosphodiesterase Activity Determinations

Phosphodiesterase activity of the preparations was determined asfollows. PDE assays utilizing a charcoal separation technique wereperformed essentially as described in Loughney et al. (1996). In thisassay, PDE activity converts [32P]cAMP or [32P]cGMP to the corresponding[32P]5′-AMP or [32P]5′-GMP in proportion to the amount of PDE activitypresent. The [32P]5′-AMP or [32P]5′-GMP then was quantitativelyconverted to free [32P]phosphate and unlabeled adenosine or guanosine bythe action of snake venom 5′-nucleotidase. Hence, the amount of[32P]phosphate liberated is proportional to enzyme activity. The assaywas performed at 30° C. in a 100 μL reaction mixture containing (finalconcentrations) 40 mM Tris HCl (pH 8.0), 1 μM ZnSO₄, 5 mM MgCl₂, and 0.1mg/mL bovine serum albumin (BSA). PDE enzyme was present in quantitiesthat yield <30% total hydrolysis of substrate (linear assay conditions).The assay was initiated by addition of substrate (1 mM [32P]cAMP orcGMP), and the mixture was incubated for 12 minutes. Seventy-five (75)μg of Crotalus atrox venom then was added, and the incubation wascontinued for 3 minutes (15 minutes total). The reaction was stopped byaddition of 200 μL of activated charcoal (25 mg/mL suspension in 0.1 MNaH₂PO₄, pH 4). After centrifugation (750×g for 3 minutes) to sedimentthe charcoal, a sample of the supernatant was taken for radioactivitydetermination in a scintillation counter and the PDE activity wascalculated.

Purification of PDE5 from S. cerevisiae

Cell pellets (29 g) were thawed on ice with an equal volume of LysisBuffer (25 mM Tris HCl, pH 8, 5 mM MgCl₂, 0.25 mM DTT, 1 mM benzamidine,and 10 μM ZnSO₄). Cells were lysed in a Microfluidizer® (MicrofluidicsCorp.) using nitrogen at 20,000 psi. The lysate was centrifuged andfiltered through 0.45 μm disposable filters. The filtrate was applied toa 150 mL column of Q SEPHAROSE® Fast-Flow (Pharmacia). The column waswashed with 1.5 volumes of Buffer A (20 mM Bis-Tris Propane, pH 6.8, 1mM MgCl₂, 0.25 mM DTT, 10 μM ZnSO₄) and eluted with a step gradient of125 mM NaCl in Buffer A followed by a linear gradient of 125-1000 mMNaCl in Buffer A. Active fractions from the linear gradient were appliedto a 180 mL hydroxyapatite column in Buffer B (20 mM Bis-Tris Propane(pH 6.8), 1 mM MgCl₂, 0.25 mM DTT, 10 μM ZnSO₄, and 250 mM KCl). Afterloading, the column was washed with 2 volumes of Buffer B and elutedwith a linear gradient of 0-125 mM potassium phosphate in Buffer B.Active fractions were pooled, precipitated with 60% ammonium sulfate,and resuspended in Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mMNaCl, 0.5 mM DTT, and 10 μM ZnSO₄). The pool was applied to a 140 mLcolumn of SEPHACRYL® S-300 HR and eluted with Buffer C. Active fractionswere diluted to 50% glycerol and stored at −20° C.

The resultant preparations were about 85% pure by SDS-PAGE. Thesepreparations had specific activities of about 3 μmol cGMP hydrolyzed perminute per milligram protein.

Inhibitory Effect on cGMP-PDE

cGMP-PDE activity of compounds of the present invention was measuredusing a one-step assay adapted from Wells et al., Biochim. Biophys.Acta, 384, 430 (1975). The reaction medium contained 50 mM Tris-HCl, pH7.5, 5 mM magnesium acetate, 250 μg/ml 5′-Nucleotidase, 1 mM EGTA, and0.15 μM 8-[H³]-cGMP. Unless otherwise indicated, the enzyme used was ahuman recombinant-PDE5 (ICOS Corp., Bothell, Wash.).

Compounds of the invention were dissolved in DMSO finally present at 2%in the assay. The incubation time was 30 minutes during which the totalsubstrate conversion did not exceed 30%.

The IC₅₀ values for the compounds examined were determined fromconcentration-response curves typically using concentrations rangingfrom 10 nM to 10 μM. Tests against other PDE enzymes using standardmethodology showed that compounds of the invention are selective for thecGMP-specific PDE enzyme.

Biological Data

The compounds according to the present invention were typically found toexhibit an IC₅₀ value of less than 500 nM. In vitro test data forrepresentative compounds of the invention is given in the followingtable:

TABLE 1 In vitro results Example PDE5 IC₅₀ (nM) 1 48.1 2 401.7 3 1.2 46.0 5 288.0 6 151.0 7 7.0 8 37.0

Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof, and, therefore, only such limitations should be imposedas are indicated by the appended claims.

1. A compound having a formula

wherein R¹ is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynyl, haloC₁₋₆alkyl, C₃₋₈cycloalkyl,heteroC₃₋₈cycloalkyl, C₃₋₈cycloalkylC₁₋₃alkyl, arylC₁₋₃alkyl, andheteroarylC₁₋₃alkyl; R² is selected from the group consisting of anoptionally substituted monocyclic aromatic ring selected from the groupconsisting of benzene, thiophene, furan, and pyridine, and an optionallysubstituted bicyclic ring

wherein the fused ring A is a 5- or 6-membered ring, saturated orpartially or fully unsaturated, and comprises carbon atoms andoptionally one or two heteroatoms selected from oxygen, sulfur, andnitrogen; R³ is selected from the group consisting of hydrogen andC₁₋₆alkyl, or R¹ and R³ together represent a 3- or 4-membered alkyl oralkenyl chain component of a 5- or 6-membered ring; R⁴, independently,is selected from the group consisting of aryl, Het, C₃₋₈cycloalkyl,YC₃₋₈cycloalkyl (wherein Y is oxygen, sulfur, or NR^(a)), C(═O)R^(a),OC(═O)R^(a), C(═O)OR^(a), C₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneHet,C₁₋₄alkyleneC(═O)OR^(a), C(═O)NR^(a)SO₂R^(c), C(═O)C₁₋₄alkyleneHet,C(═O)NR^(a)R^(b), C(═O)NR^(a)R^(c), C(═O)NR^(a)C₁₋₄alkyleneOR^(b),C(═O)NR^(a)C₁₋₄alkyleneHet, OR^(a), OC₁₋₄alkyleneC(═O)OR^(a),OC₂₋₄alkyleneNR^(a)R^(b), OC₁₋₄alkyleneHet, OC₂₋₄alkyleneOR^(a),OC₁₋₄alkyleneNR^(a)C(═O)OR^(b), NR^(a)R^(b),NR^(a)C₁₋₄alkyleneNR^(a)R^(b), NR^(a)C(═O)R^(b), NR^(a)C(═O)NR^(a)R^(b),N(SO₂C₁₋₄alkyl)₂, NR^(a)(SO₂C₁₋₄alkyl), nitro (NO₂), trifluoromethyl,trifluoromethoxy, cyano (CN), SO₂NR^(a)R^(b), SO₂R^(a), SOR^(a), SR^(a),and OSO₂CF₃; R⁵ is selected from the group consisting of hydrogen,halogen, and C₁₋₆alkyl; or R⁴ and R⁵ are taken together with the carbonatoms to which they are attached to form a 5-, 6-, or 7-membered ring,saturated or partially or fully unsaturated, optionally substituted andoptionally containing one or two heteroatoms selected from the groupconsisting of oxygen, sulfur, and nitrogen; R^(a) is selected from thegroup consisting of hydrogen, C₁₋₆alkyl, C₃₋₈cycloalkyl, aryl,arylC₁₋₃-alkyl, C₁₋₃alkylenearyl, and Het; R^(b) is selected from thegroup consisting of hydrogen, C₁₋₆alkyl, aryl, arylC₁₋₃alkyl,C₁₋₃alkylenearyl, and Het; R^(c) is phenyl or C₄₋₆cycloalkyl, eitheroptionally substituted with one or more substituent selected from thegroup consisting of halo, C(═O)OR^(a), and OR^(a); Het is a 5- or6-membered heterocyclic group, saturated or partially or fullyunsaturated, containing at least one heteroatom selected from the groupconsisting of oxygen, nitrogen, and sulfur, and optionally substitutedwith C₁₋₄alkyl or C(═O)OR^(b); q is 1, 2, or 3; and pharmaceuticallyacceptable salts and hydrates thereof.
 2. The compound of claim 1represented by the formula

and pharmaceutically acceptable salts and solvates thereof.
 3. Thecompound of claim 1 wherein R¹ is selected from the group consisting ofhydrogen, C₁₋₄alkyl, haloC₁₋₄alkyl, C₃₋₆cycloalkyl, andC₃₋₆cycloalkylmethyl.
 4. The compound of claim 1 wherein R³ is hydrogen.5. The compound of claim 1 wherein R² is an optionally substitutedbicyclic ring selected from the group consisting of naphthalene, indene,benzoxazole, benzothiazole, benzisoxazole, benzimidazole, quinoline,indole, benzothiophene, and benzofuran.
 6. The compound of claim 1wherein R² is

and wherein n is an integer 1 or 2, and X, independently, are C(R^(a))₂,O, S, or NR^(a).
 7. The compound of claim 1 wherein R², substituted orunsubstituted, is selected from the group consisting of


8. The compound of claim 7 wherein R² is substituted with a substituentselected from the group consisting of halogen, C₁₋₃alkyl, OR^(a),CO₂R^(a), halomethyl, halomethoxy, cyano, nitro, and NR^(a)R^(b).
 9. Thecompound of claim 1 wherein R⁴ is selected from the group consisting ofaryl, trifluoromethyl, trifluoromethoxy, C(═O)R^(a), C(═O)OR^(a),C(═O)NR^(a)R^(b), C(═O)NR^(a)R^(c), OR^(a), CN, C₁₋₄alkyleneNR^(a)R^(b),OC₂₋₄alkyleneNR^(a)R^(b), SO₂NR^(a)R^(b), OC(═O)R^(a), NR^(a)R^(b), Het,C₃₋₈-cycloalkyl, and YC₃₋₈cycloalkyl.
 10. The compound of claim 9wherein R⁴ is selected from the group consisting of CH₂NR^(a)R^(b),aryl, CN, OR^(a), C(═O)OR^(a), and NR^(a)R^(b).
 11. The compound ofclaim 10 wherein R⁴ is selected from the group consisting of CH₂NH₂,CO₂H, CO₂CH₃, C₆H₅, OCH₂C₆H₅, OH, CN, and OCH₃.
 12. The compound ofclaim 1 wherein R⁴ and R⁵ are taken together to form a 6-memberedsaturated or unsaturated ring, optionally substituted and optionallycontaining one or two heteroatoms.
 13. The compound of claim 12 whereinR⁴ and R⁵ are taken together to form a phenyl ring.
 14. The compound ofclaim 1 wherein R⁵ is hydrogen.
 15. A compound selected from the groupconsisting of

and pharmaceutically acceptable salts and solvates thereof.
 16. Apharmaceutical composition comprising a compound of claim 1, togetherwith a pharmaceutically acceptable diluent or carrier.
 17. A method oftreating a male animal in the treatment of male erectile dysfunctioncomprising treating said male animal with an effective amount of apharmaceutical composition comprising a compound of claim 1, togetherwith a pharmaceutically acceptable diluent or carrier.
 18. The method ofclaim 17 wherein the treatment is an oral treatment.
 19. A method oftreating a male or female animal in the treatment of a condition whereinhibition of a cGMP-specific PDE is of a therapeutic benefit comprisingtreating said animal with an effective amount of a pharmaceuticalcomposition comprising a compound of claim 1, together with apharmaceutically acceptable diluent or carrier, wherein the condition isselected from the group consisting of stable angina, unstable angina,variant angina, hypertension, pulmonary hypertension, chronicobstructive pulmonary disease, malignant hypertension, pheochromocytoma,acute respiratory distress syndrome, congestive heart failure, acuterenal failure, chronic renal failure, atherosclerosis, a condition ofreduced blood vessel patency, a peripheral vascular disease, a vasculardisorder, thrombocythemia, myocardial infarction, bronchitis, chronicasthma, allergic asthma, allergic rhinitis, glaucoma, peptic ulcer, agut motility disorder, postpercutaneous transluminal coronaryangioplasty, carotid angioplasty, post-bypass surgery graft stenosis,osteoporosis, preterm labor, benign prostatic hypertrophy, and irritablebowel syndrome.
 20. A method of treating a female animal in thetreatment of female sexual arousal disorder comprising treating saidfemale animal with an effective amount of a pharmaceutical compositioncomprising a compound of claim 1, together with a pharmaceuticallyacceptable diluent or carrier.
 21. The method of claim 20 wherein thetreatment is an oral treatment.